Marked peptides having affinity for a phospholipid and uses

ABSTRACT

The present invention relates to a peptide labelled with fluorine-18 for the specific recognition of lipid vectors. The peptide of the invention comprises the following peptide sequence (PI):  
                     J 1 -J 2 -J 3 -J 4 -J 5 -J 6 -Z 7 -U 8 -J 9 -J 10 -U 11 -Arg-J 13 -J 14 -         U 15 -Lys-Gly-X 18 -Gly-Thr-J 21 -Glu-J 23 -J 24 -U 25 -J 26 -     J 27 -J 28 -U 29 -J 30 -J 31 -Arg-J 33 -J 34 -J 35 -J 36 -B 37 -J 38 -     J 39 -U 40 -J 41 -J 42 -J 43 -U 44 -J 45 -J 46 -J 47 -J 48 -J 49 -Arg-     J 51 -U 52 -J 53 -J 54 -Asp-U 56 -Lys-Ser-Z 59 -Leu-J 61 -J 62 -     J 63 -J 64 -Z 65 -J 66 -J 67 -U 68 -J 69 -J 70 -J 71 -U 72 -J 73 -J 74 -     J 75  (I)                    
 
in which the amino acids J are chosen independently of each other from natural amino acids, or derivatives thereof, in such a manner that at least 50% of them are polar residues chosen from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Lys, Orn, Pro, Ser, Thr and Tyr, the amino acids U are chosen from Ala, Cys, Gly, Ile, Leu, Met, Phe, Trp, Tyr and Val, the amino acid X 18  is chosen independently of the other amino acids of the sequence from Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Ser, Thr, Trp, Tyr and Val, the amino acid B 37  is chosen independently of the other amino acids of the sequence from Arg, Ala, Cys, Gly, Ile, Leu, Met, Phe, Trp, Tyr and Val, the amino acid Z 7  is chosen independently of the other amino acids from Asp and Glu, the amino acids Z 59  and Z 65  are chosen independently from Glu, Asp, Lys and Arg, the superscripts of the residues J, Z, U, X and B representing the positions of these amino acids in the said sequence.

TECHNICAL FIELD

The present invention relates to a family of peptides labelled withfluorine-18 which have enhanced affinities for phospholipids, and totheir uses.

In general, the peptides of the present invention are useful for thespecific recognition of lipid molecules. They can be used forengineering and creating compounds recognizing and sequestering lipids,in particular negatively charged lipids, such as phosphatidylserines,phosphatidic and lysophosphatidic acids, phosphatidylglycerols,cardiolipins and sphingosine-1-phosphates.

The abovementioned lipids play an important role, in particular in cellsignalling and may be present at the outer surface of cell membranesand/cr circulate in the blood stream following a wide variety ofpathological events.

Various cellular events result in the appearance of negatively chargedlipids and in particular of phosphatidylserines (PS) at the outersurface of cells; these events can result either from a fortuitous orpathological impairment of the cell, or from a programmed cellular eventsuch as cell death or apoptosis. The appearance of PS at the outersurface of cells therefore constitutes an important “primary message”indicating the existence of a dysfunction. In the case of the bloodclotting process, the mechanism is well described: the impairment of theendothelial cells of the blood vessels, either for accidental reasons,or for more complex pathological reasons, causes the appearance of thisPS message at the outer surface of the cells in contact with the bloodstream. This message is immediately recognized by certain circulatingproteins which then trigger a cascade of events resulting in thewell-known phenomenon of blood clotting.

The invention exploits the property of the labelled peptides, which itprovides, to bind, in the presence or absence of calcium, to lipids andin particular to those which are negatively charged, for the developmentof compounds which can be used as research and diagnostic tools in thefield of the recognition of lipid effectors and of the detection ofapoptosis, of blood clotting disorders, of septic shock and of acuteinflammatory pathologies in particular.

The labelled peptides of the invention are coupled to a radioactivehalogen, a positron emitter, which is fluorine ¹⁸F. With these labelledpeptides, it is therefore possible, for example to detect apoptoticcells and to recognize negatively charged membrane microdomains.

They can be used for “in vitro” detection of pathologies involving theappearance of centres exposing negatively charged lipids at the surfaceof the cells and/or the release of microvesicles into the blood.

The labelled peptides of the present invention may also be used for thein vivo detection and the imaging of apoptotic foci, of thromboticregions, and in general of centres exposing negatively charged lipids atthe surface of cells and/or the release of microvesicles into the blood,for example by means of scintigraphic images, acquired by positronemission tomography (PET).

Other applications will also appear to persons skilled in the art onreading the description which follows.

STATE OF THE ART

A family of proteins, called annexins, have been described in the priorart as presenting a reversible functional anchor to the cell membrane,regulated by the calcium concentration and the presence of anionicphospholipids. Annexins constitute a family of proteins expressed in awide variety of tissues, both in animals and in plants. It appears thatthey are not expressed either in bacteria or in yeast.

The structure of annexins comprises four domains of about 70 aminoacids, or residues, with a very slight sequence homology, but withpractically identical topology.

In the document WO 92/19279, J. TAIT describes conjugates havingaffinity for phospholipids. It describes in particular the use of anannexin, in particular of annexin V, to manufacture an active conjugatewhich can be used as thrombolytic agent.

Unfortunately, the compound described in this document and prepared fromwhole annexin by a method of genetic recombination has manydisadvantages which are in particular a low yield and a high cost ofmanufacture. The major disadvantages are especially the production of afragile conjugate because of its complex topology resulting inirreversible unfolding. In addition, these molecules exhibit a majortoxicity for the kidney and the heart.

The present inventors have described, in application WO-A-00/20453, afirst family of peptides overcoming the abovementioned disadvantages andexhibiting affinity for phospholipids and enhanced stability.

Moreover, it is known that for use in research and diagnosis,macromolecules, such as proteins or peptides, can be coupled to alabelling molecule allowing their detection, this labelling molecule maybe for example a fluorescent molecule, gold particles, a paramagneticcompound or a molecule bearing a radioelement.

Proteins have been radioactively labelled with radioisotopes, iodine andvarious radioisotopes of metals, such as technetium, indium and gallium.More recently, proteins have been labelled with fluorine-18.

For example, peptides coupled to radioelements, such as fluorine, allow“in vivo” detection of the localization of thrombotic regions during allsorts of stroke, in particular of apoptotic and inflammatory foci, usingimaging systems.

Thus, radioactive atoms which emit positrons having a short life span,and especially ¹⁸F, can in particular be detected by positron emissiontomography (PET) apparatus.

Radioactive labelling with fluorine-18 poses, in particular because ofthe very short life span of fluorine-18 (close to 109.8 minutes),specific problems which are such that labelling with fluorine-18 isbasically different from that with other halogens, such as iodine.

The abovementioned labelling may be carried out by any of theconventional techniques of organic chemistry known to persons skilled inthe art, and by the synthesis of protein and peptide markers bearing oneor more radioactive atoms with a short life span, in particular ¹⁸F.This marker generally consists, on the one hand, of a part capable ofreceiving, for example, an atom of ¹⁸F and, on the other hand, of a partcontaining any conventional functional group for linking to themacromolecule, for example to the protein.

These markers must satisfy the requirement for rapid and easy synthesis,because due to the short life span of radioisotopes such as ¹⁸F, theduration of synthesis should generally not exceed a few hours.

In addition, this synthesis, because of the high radioactivity of thecompounds used, must be capable of being carried out by automated means.

Thus, the methods for labelling proteins or peptides with fluorine-18involve markers also called labelled “conjugates” or “synthons”, whichare classified into three main families, depending on whether they reactwith the amine groups, the sulphydryl groups, or the carbohydrate groupsof the macromolecules, such as proteins and peptides.

Among the compounds or conjugates reacting with amino groups, there maybe mentioned imidates, such as 3-[¹⁸F]fluoro-5-nitrobenzoimidate, whichreact, for example, with the ε-NH₂ group of lysine in order to bind to aprotein; activated esters, such as N-succinimidyl-[¹⁸F]fluorobenzoate;carboxylic acids, such as N-(4-[¹⁸F]fluorobenzoic) acid; aldehydes, suchas 4-[¹⁸F]pentafluorobenzaldehyde and isothiocyanates, such as 4-([¹⁸F]fluoromethylphenylisothiocyanate).

Activated halides, such as (4-[¹⁹F]fluorophenacyl)bromide, react withthe amino groups, such as the ε-NH₂ group of lysine and the —SH group ofcysteine.

Amines, such as 1-(4-([¹⁸F]fluoromethyl)benzoyl)aminobutane-4-amine,react with the CO₂H groups, for example of glutamic acid or of asparticacid or with the CHO groups of glycoproteins.

Nitrenes with photochemical active centres, such as azidophenacyl[¹⁸F]fluoride, also react with the amino groups, for example the ε-NH₂group of lysine.

The most effective and most widely described method for labellingproteins and peptides is that which uses activated acids, but it is alsothe method which exhibits the greatest nonspecificity because all thenucleophilic sites of the amino acids of the proteins or peptides willreact with the labelled marker, conjugate or synthon.

Two methods which are more specific for labelling peptides andnucleotides exhibit good specificity toward the sulphur atoms, forexample, of cysteine for peptides and for a phosphorothioate functionalgroup for nucleotides.

They include, first of all, methods using haloacetamide “synthons”which, although satisfactory, have the disadvantage of being very slowand therefore not very suitable for ¹⁸F, because of its life span.

They then include methods using activated maleimides which can bind tothe SH groups with a very good specificity because the reaction is veryslow in relation, for example, to the ε-NH₂ sites of lysine.

The reaction scheme involving the maleimido group is the following, inthe case of a protein:

in which X represents —S—.

For any labelling, regardless of the type, molecules comprising amaleimide radical are currently considered as being the best, as regardstheir reactivity with macromolecules, such as peptides or proteins.

The document by SHIUE C.-Y. et al., J. Label Compounds Radiopharm 26:278-280 (1988), describes the compounds:

The first of these compounds is not easy to label with fluorine-18 at ahigh specific activity.

Indeed, only fluorine F₂ would allow easy labelling of the “iodine type”and it happens to be the case precisely that F₂ is generally a productwith a low specific activity.

In particular F₂ is not suitable for the manufacture of so-called“radiotracer” compounds which are preferably aimed at according to theinvention quite simply because the injected mass of labelled moleculebecomes too large and in that case the basic principle guiding this“tracer”, namely the extremely easy occupation (for example less than5%) of the receptor sites, is not satisfied.

In addition, the synthesis of the first of these compounds is difficult;it is indeed carried out in four stages requiring a long period withvery low yields, and relatively complex chemical conversions. Thismethod cannot therefore be easily automated.

The second of the compounds cited in the SHIUE et al. document containsan amide chain which is not chemically very strong and which is easilycleaved or broken in vivo.

Its use for diagnostic applications cannot therefore be envisaged. Inaddition the synthesis of this second compound comprises three stagesand the final yield is low, for example close to 10% (“EOB” “End ofBombardment”).

The document U.S. Pat. No. 4,735,792 relates to molecules of formula:

in which X is a radioactive halogen chosen from bromine-75, bromine-76,bromine-82, iodine-123, iodine-125, iodine-131 and fluorine-18.

However, only the molecule labelled with iodine-125 is effectivelyprepared.

The preparation of a molecule labelled with fluorine-18 is not mentionedor evoked, and the remarks already made above in the case of the firstcompound of the SHIUE et al., document also apply in the case of thedocument U.S. Pat. No. 4,735,792.

Persons skilled in the art, on reading this document, possess noinformation allowing them to specifically prepare a compound labelledwith fluorine-18 and if they envisage doing it, they would use F₂ andwould thus arrive at a compound with a low specific activity, which isunusable in “PET” imaging.

It can be considered additionally that the chemistry used to manufacturethe fluorinated compound of the document U.S. Pat. No. 4,735,792 is acomplex and long chemistry.

DISCLOSURE OF THE INVENTION

The aim of the present invention is precisely to provide a novel familyof peptides, labelled with a radioactive halogen which is fluorine ¹⁸Fusing a novel labelling compound, the peptide having affinity forlipids, in particular for phospholipids, more specific and furtherimproved compared with the prior art products, and the labellingcompound having, inter alia, high reactivity, high selectivity inparticular towards sulphur atoms such as those of the thiol functionalgroups of cysteines, and a good specific activity and it being possiblefor the said labelling compound in addition to be manufactured by amethod which is simple, reliable, easily automatible, rapid and of shortduration.

The peptides of the invention have in addition the advantages of beingchemically more stable than the prior art compounds and of being able tobe manufactured reproducibly, with a high yield and a very low cost ofproduction compared with the prior art compounds.

Fluorine-18 (¹⁸F) is a positron emitter which allows detection, by meansof the labelled peptides of the present invention, of negatively chargedlipids in any region of the body by positron (PET) cameras. Thiscoupling of the peptides of the present invention to ¹⁸F makes itpossible for example to detect, with a resolution better than themillimetre range, the presence of cells exhibiting phosphatidylserine(PS), present at the outer surface of the cells involved inphysiopathological processes such as programmed cell death, apoptosis,blood clotting, inflammatory reaction in vivo in any living being. Italso allows such a detection in vitro in laboratory tests.

These labelled peptides of the present invention also make it possibleto precisely quantify for example the number of cells havingphosphatidylserine.

The peptides of the present invention are characterized in that theycomprise the following peptide sequence (PI):J¹-J²-J³-J⁴-J⁵-J⁶-Z⁷-U⁸-J⁹-J¹⁰-U¹¹-Arg-J¹³-J¹⁴-U¹⁵-Lys-Gly-X¹⁸-Gly-Thr-J²¹-Glu-J²³-J²⁴-U²⁵-J²⁶-J²⁷-J²⁸-U²⁹-J³⁰-J³¹-Arg-J³³-J³⁴-J³⁵-J³⁶-B³⁷-J³⁸-J³⁹-U⁴⁰-J⁴¹-J⁴²-J⁴³-U⁴⁴-J⁴⁵-J⁴⁶-J⁴⁷-J⁴⁸-J⁴⁹-Arg-J⁵¹-U⁵²-J⁵³-J⁵⁴-Asp-U⁵⁶-Lys-Ser-Z⁵⁹-Leu-J⁶¹-J⁶²-J⁶³-J⁶⁴-Z⁶⁵-J⁶⁶-J⁶⁷-U⁶⁸-J⁶⁹-J⁷⁰-J⁷¹-U⁷²-J⁷³-J⁷⁴- J⁷⁵ (PI)in which J, Z, U, X and B represent amino acids such that:

-   -   the amino acids J are chosen independently of each other from        natural amino acids, or derivatives thereof, in such a manner        that at least 50% of them are polar residues chosen from Arg,        Asn, Asp, Cys, Gln, Glu, Gly, His, Lys, Orn, Pro, Ser, Thr and        Tyr,    -   the amino acids U are chosen from Ala, Cys, Gly, Ile, Leu, Met,        Phe, Trp, Tyr and Val,    -   the amino acid X¹⁸ is chosen independently of the other amino        acids of the sequence from Ala, Asn, Cys, Gln, Gly, His, Ile,        Leu, Met, Phe, Ser, Thr, Trp, Tyr and Val,    -   the amino acid B³⁷ is chosen independently of the other amino        acids of the sequence from Arg, Ala, Cys, Gly, Ile, Leu, Met,        Phe, Trp, Tyr and Val,    -   the amino acid Z⁷ is chosen independently of the other amino        acids of the sequence from Asp and Glu,    -   the amino acids Z⁵⁹ and Z⁶⁵ are chosen independently from Glu,        Asp, Lys and Arg,        the superscripts of J, Z, U, X and B representing the positions        of these amino acids in the said sequence.

According to the invention, these peptides of the present invention, asdefined above, are labelled directly or indirectly with a labellingcompound of the present invention of the following general formula (CI):

in which:

-   -   m represents an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5,        6, 7, 8, 9 or 10;    -   n represents an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5,        6, 7, 8, 9 or 10;    -   Y represents a group chosen from alkyl groups, monocyclic or        bicyclic heterocyclic groups chosen from imidazolyl, pyrazolyl,        benzimidazolyl, pyridinyl, piridazinyl, pyrimidinyl, pyrazinyl,        triazinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl,        quinoxalinyl and purinyl groups, it being possible for Y to be        optionally substituted with one or more substituents, each of        these substituents being chosen independently from hydrogen,        (nonradioactive) halogens, phenyl, C₁₋₆ alkyl, C₁₋₆ alkoxy,        aryloxy, amino, mono- or di(C₁₋₆ alkyl)amino, mono- or        di(aryl)amino, thio, C₁₋₆ alkylthio, arylthio, formyl, C₁₋₆        alkylcarbonyl, arylcarbonyl, carbonyl, C₁₋₆ alkoxycarbonyl,        aryloxycarbonyl, C₁₋₆ alkylaminocarbony arylaminocarbonyl and        trifluoromethyl groups;    -   β represents a radical of formula:        (γ)_(a)-((CR₁R₂)_(b)—(V)_(c))_(d)—( (CR₃R₄)_(e)—(W)_(f))_(g)—        in which:    -   a, b, c, d, e, f, g each independently represent an integer from        0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9;    -   γ, V and W each independently represent —NR—₁, —O—, —S—,        ethynyl, —CR₁═CR₂, —(C═O)—, —(C═S)—, —C(═NR₁)—, —C(═O)O—,        —(C═S)S—, —C(═NR₁)NR₂—, —CR₁R₂—, —CR₁OR₂—, —CR₁NR₂R₃—, where R₁,        R₂: R₃ and R₄ are independently chosen from hydrogen, halogens,        phenyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, aryloxy, amino, mono- or        di(C₁₋₆ alkyl)amino, mono- or di(aryl)amino, thio, C₁₋₆        alkylthio, arylthio, formyl, C₁₋₆ alkylcarbonyl, arylcarbonyl,        carbonyl (C₁₋₆)alkoxycarbonyl, aryloxycarbonyl, C₁₋₆        alkylaminocarbonyl, arylaminocarbonyl and trifluoromethyl        groups.

Generally, in the present description, halogen means fluorine, chlorine,bromine or iodine. C₁₋₆ alkyl corresponds to linear and branched chainsaturated hydrocarbon radicals having from 1 to 6 carbon atoms, such asmethyl, ethyl, propyl, butyl, pentyl and hexyl.

The attachment and the substitution of the heterocycles, aryl group, andthe like, may be made in any position.

Likewise, the attachment of the ¹⁸F to Y or β may be made in anyposition, in particular to any position on a heterocycle.

The compounds according to the present invention are basicallydistinguishable from the prior art compounds because of their specificstructure in which the part bearing the fluorine-18 atom consists,according to the invention, of a specific group Y which is in particulara pyridinyl group; the part for linking, coupling to the peptideconsists, according to the invention, of a specific functional group,namely a maleimido functional group; and, finally, the part for bindingto the peptide and the part bearing the fluorine-18 atom are linkedaccording to the invention by a spacer chain or arm which is alsospecific, for example of the type comprising alkyl (generally from 2 to6C), alkyl ether, phenylalkyl ethers, alkenyl, which are not fragile andare not susceptible to breakings “in vivo”.

The expression direct labelling is understood to mean a direct coupling,without intermediate, such as a spacer arm, of the labelling compound(CI) with the peptide of the present invention, for example by means ofa free —SH functional group of the peptide defined above; this may be inparticular the thiol functional group of a cysteine of the peptide.

This coupling of the labelling compound (CI) with the peptide can becarried out either on the sequence (PI) defined above, for example oncysteine residues localized at the surface of the protein, but in amanner which is not disruptive for the functional groups for bindingcalcium and phospholipids, or on a portion of the peptide other thanthat of the said sequence (PI). The coupling occurs through themaleimide functional group of the compound (CI).

More precisely, the said coupling is achieved by the reaction of thedouble bond of the maleimido group of the compound according to theinvention with specifically an —SH (thiol) functional group of acysteine forming part of the peptide.

One of the advantages linked to the specific structure of the compoundsaccording to the invention is to allow specific, or even exclusive,labelling of the cysteines, whereas most of the other “synthons” onlyallow nonspecific labelling of the lysines and of the cysteines.

The selective, or even exclusive, labelling of the cysteines is due tothe presence, in the labelling molecule of the invention, of a“dedicated” functional group, namely the maleimido functional group,which is a dedicated functional group for the chemoselectivity towardsthe thiols of the cysteines.

The expression indirect labelling is understood to mean the use of aspacer arm linked, on the one hand, to the labelling compound, and, onthe other hand, to the peptide as defined above. This spacer arm mayhave the role of putting the marker and the peptide apart so that nosteric hindrance prevents the peptide from recognizing its target(negatively charged lipid). This spacer arm may be of an organic nature,for example an alkyl provided with a thiol group, or a peptide sequencecomprising a cysteine, for example -(Gly)_(n)-Cys where n is equal to orgreater than 1.

It is evident that the coupling of the labelling compound with thepeptide in accordance with the present invention will be in any casesuch that it does not inhibit or inhibits in a manner which is not verydisruptive the activity for the specific recognition of the negativelycharged lipids by the peptide of the present invention.

The above peptide sequence (PI) falls in space in order to adopt itstertiary conformation which is the active form of the peptide.

The amino acids 12, 15, 16, 17, 19, 20, 22, 50, 55, 57, 58, 59, 60 and65 of the peptide (PI) of the present invention are amino acids, orresidues, involved directly or indirectly in the binding to lipids, thatis to say that they are involved either in the three-dimensionalstructure of the peptide so that it adopts its active conformation forrecognition, or in the site for recognition of the lipid.

The amino acids J are the surface amino acids or residues of thispeptide when it is in its folded and active conformation. These residuesare arranged in space such that they are partially or completely exposedto the solvent. According to the present invention, these amino acids Jmay for example be chosen independently of each other from all thenatural amino acid residues Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Orn, Phe, Pro, Ser, Thr, Trp, Tyr, and Val: and insuch a manner that at least 50% of them are polar residues chosen fromArg, Asn, Asp, Cys, Gln, Glu, Gly, His, Lys, Orn, Pro, Ser and Thr.Examples are given in the appended sequence listing.

The amino acids U are the core residues of this peptide. In the foldedand active conformation of the peptide, they are arranged in space closeto each other and not exposed to the solvent. They constitute thehydrophobic core of the protein. The compact assembly of the atoms ofthese residues plays a predominant role for the stability of the peptidein its active conformation. These residues may be chosen from the listof amino acids U described above. Various examples of combinations ofcore residues in the peptide sequence (PI) of the present invention aregiven in the table (1) below: TABLE 1 U⁸ U¹¹ U¹⁵ U²⁵ U²⁹ B³⁷ U⁴⁰ U⁴⁴ U⁵²U⁵⁶ U⁶⁸ U⁷² Ex a) Val Leu Met Ile Leu Arg Ile Tyr Leu Leu Val Leu Ex b)Ala Ile Ile Ile Leu Arg Ile Tyr Leu Leu Ile Leu Ex c) Ala Ile Ile IleLeu Arg Ile Tyr Leu Leu Met Val Ex d) Ala Leu Met Leu Leu Arg Ile TyrLeu Leu Ile Met Ex e) Ala Leu Met Ile Ile Arg Val Tyr Leu Leu Ile Met Exf) Ala Leu Met Ile Ile Arg Ile Phe Leu Leu Ile Met Ex g) Ala Leu Met IleVal Arg Ile Phe Leu Leu Ile Phe Ex h) Val Leu Met Ile Leu Arg Ile PheLeu Leu Ile Met Ex i) Ala Leu Met Ile Leu Arg Ile Phe Leu Leu Ile Met Exj) Ala Leu Met Ile Leu Arg Ile Tyr Leu Leu Ala Ala Ex k) Val Leu Met IleLeu Arg Ile Tyr Leu Leu Val Leu Ex l) Val Leu Met Ile Leu Arg Ile PheLeu Leu Val Leu(Ex = exemple)(Ex=Example)

The residue X¹⁸ has the role of maintaining the structure of theGly-X-Gly loop in the active form of the peptide, in particular wherethe residues Z⁵⁹ and Z⁶⁵ are Glu, to modulate the hydrophobic andlipophilic character of this loop, and to optionally provide specificnovel interactions with phospholipids. That is the case for example forthe residues Asn, Cys, Ser, Thr, Trp and Tyr.

The residues Z⁵⁹ and Z⁶⁵ may be advantageously lysine residues, whichhas the effect of replacing the calcium ion with the positively chargedgroup —NH₃ ⁺ of the lysine and of enhancing the affinity of the peptidefor a negatively charged membrane.

The peptide (PI) of the present invention, in its active form, comprisesthree sites for binding to a calcium ion where the calcium ion complexedby this site constitutes one of the ligands of a negatively chargedphospholipid. The first of these sites, called principal site, involvesthe residues 15, 18, 19 and 59 as calcium ligands. The second of thesesites, called secondary site, involves the residues 20 and 22 as calciumligands. The third of these sites, which is a secondary site of lowaffinity involves the residues 57, 60 and 65 as calcium ligands.

The residues which are involved overall in the binding to thephospholipids are the residues 12, 15, 16, 19, 20, 22, 50, 55, 57, 58,59, 60 and 65. This list includes residues involved in the bindings ofcalcium, the phospholipids being calcium ligands.

These residues may of course be replaced by residues playing the samerole for the purpose of the same result in accordance with the presentinvention.

By way of example, according to the invention, the peptide of formula(PI) may be advantageously a peptide sequence chosen from the appendedpeptide sequences ID No. 1 to ID No. 10.

The sequence (PI) represents the peptides of the present invention intheir shortest functional form. It is of course understood that thissequence may additionally comprise, linked to the N-terminal end and/orto the C-terminal end of the sequence (PI), one or more amino acids, forexample from 1 to 15 amino acids, in general from 1 to 10 amino acids.Most preferably, these additional amino acids do not modify or onlyslightly modify the activity of the peptides, or else enhance it.

For example, a small sequence, called below functionalization sequencemay be useful in particular for attaching a marker to the peptide, forattaching a molecule for treating diseases to the peptide and/or forattaching the said peptide to a support. The length of thisfunctionalization sequence may be adapted according to its use. Ofcourse, the latter will preferably not interfere with the activity ofthe peptide of the present invention. Persons skilled in the art willeasily know how to adapt the length and the nature of thisfunctionalization sequence according to the use that will be made of apeptide of the present invention.

Thus, according to a first particular embodiment of the presentinvention, the peptides of the present invention may contain, forexample at the N-terminal end, a functionalization sequence of threeamino acids. This functionalization sequence allows direct attachment ofthe labelling compound (CI) to the peptide. The peptides in accordancewith this embodiment may be defined by the following sequence (PII):J⁻²J⁻¹-J⁰J¹-J²-J³-J⁴-J⁵-J⁶-Z⁷-U⁸-J⁹-J¹⁰-U¹¹-Arg-J¹³-J¹⁴-U¹⁵-Lys-Gly-X¹⁸-Gly-Thr-J²¹-Glu-J²³-J²⁴-U²⁵-J²⁶-J²⁷-J²⁸-U²⁹-J³⁰-J³¹-Arg-J³³-J³⁴-J³⁵-J³⁶-B³⁷-J³⁸-J³⁹-U⁴⁰-J⁴¹-J⁴²-J⁴³-U⁴⁴-J⁴⁵-J⁴⁶-J⁴⁷-J⁴⁸-J⁴⁹-Arg-J⁵¹-U⁵²-J⁵³-J⁵⁴-Asp-U⁵⁶-Lys-Ser-Z⁵⁹-Leu-J⁶¹-J⁶²-J⁶³-J⁶⁴-Z⁶⁵-J⁶⁶-J⁶⁷-U⁶⁸-J⁶⁹-J⁷⁰-J⁷¹-U⁷²- J⁷³-J⁷⁴-J⁷⁵ (PII)in which J, Z, U, X and B are as defined above.

For example, J⁻² may be Gly, J⁻¹ may be Ser or Cys and J⁰ may be Cys,Thr, Pro, Ser or Gln, preferably J⁰ is Cys.

This sequence J⁻²J⁻¹-J⁰ may be chosen for example from Gly-Ser-Cys-, andGly-Cys-Ser-. Thus, for example, each of the sequences ID No. 1 to IDNo. 10 mentioned above may contain according to choice each of theabovementioned functional sequences. The sequence ID No. 12 of theappended sequence listing is only a nonlimiting example of a sequence(PII) according to the present invention containing at its N-terminalend a functional sequence of three amino acids.

According to a second particular embodiment of the present invention,the peptide sequence (PI) may contain, for example at their N-terminalend, a functionalization sequence of four amino acids J⁻³-J⁻²J⁻¹-J⁰chosen from Gly-Ser-Gly-Cys-, Gly-Cys-Gly-Ser, and Gly-Cys-Gly-Cys. Thisfunctionalization sequence is useful for example for direct attachmentof the labelling compound (CI) to the peptide. Thus, for example, eachof the abovementioned sequences ID No. 1 to ID No. 10 may containaccording to choice each of the abovementioned functional sequences. Thesequences ID No. 11 of the appended sequence listing (several sequencesare grouped under a single name ID No. 11) are only nonlimiting examplesof sequences (PI) according to the present invention containing at itsN-terminal end a functional sequence of four amino acids.

According to a third particular embodiment of the present invention, thepeptide sequences (PI) may contain, for example at their N-terminal end,a functionalization sequence of seven to eleven amino acids. Thisfunctionalization sequence is also useful for directly attaching thecompound (CI) to the peptide. This embodiment is set out below. Thus,for example, each of the sequences ID No. 1 to ID No. 10 mentioned abovemay contain according to choice each of the abovementioned functionalsequences. It is also possible to replace the sequence Gly-Ser-Gly-Cysof the sequences ID No. 11 to 14 by Gly-Bb1-Gly-Bb2, in which Bb1 andBb2 are independently Cys or Ser. The sequences ID No. 13 and 14 of theappended sequence listing (several sequences are grouped into one underthe name ID No. 13 or 14) are merely nonlimiting examples of suchpeptides.

The peptides of the present invention have a sufficient affinity forcalcium and are capable of reversibly binding to lipid effectors, and inparticular to those which are negatively charged, such asphosphatidylserines, phosphatidic acids, phosphatidylethanolamines,phosphatidylglycerols, cardiolipins and phosphatidylinositolphosphates.

This is a family of peptides whose main property is to specificallyrecognize the appearance of lipid signals at the surface of cellmembranes in relation to the normal or pathological function of thetissues.

The peptides of the present invention may be synthesized by conventionalmethods of organic chemistry synthesis or of protein chemistry, and bygenetic recombination in vivo or in vitro, by genetic engineering, andthe like.

The peptide according to the invention may be synthesized by solid-phasechemical synthesis of the said peptide. This chemical synthesis may becarried out for example with an automatic peptide synthesizer of theApplied Biosystems, mod. 433A type. It may be carried out for example byFmoc chemistry which uses the fluorenylmethyloxycarbonyl group for thetemporary protection of the α-amino functional group of amino acids.

The technical elements for the implementation of this method of peptidesynthesis are known to persons skilled in the art. They are describedfor example in the manual Solid-Phase Organic Synthesis by Kevin Burgess(Editor) Wiley-Interscience; ISBN: 0471318256; (February 2000).

The peptide of the invention may also be manufactured by geneticrecombination in vivo for example by means of a method comprising thefollowing steps:

-   -   a) preparation of a cDNA comprising a basic sequence encoding        the said peptide    -   b) insertion of the said cDNA into an appropriate expression        vector,    -   c) transformation of an appropriate host cell with the said        vector into which the cDNA has been inserted, for replication of        the plasmid,    -   d) manufacture of the said peptide by translation of the said        cDNA in the said host cell, and    -   e) recovery of the peptide synthesized.

According to the invention, the appropriate expression vector and thehost cell are chosen according to the usual techniques for geneticrecombination. The vector may be any of the plasmids generally used inthis technique, for example a plasmid such as the vector pGEX-2T.Likewise, the cell may be chosen according to the usual techniques; itmay be for example E. coli.

When a genetic recombination technique in vitro is used, steps c) and d)of the above method are replaced respectively by steps c′) forintroducing the vector into which the cDNA has been inserted in asuitable reaction medium for replication of the plasmid, and d′) formanufacture of the said peptide by translation of the said cDNA in thesaid suitable reaction medium. The document Jagus, R. and Beckler, G. S.(1998) Overview of eukaryotic in vitro translation and expressionsystems, Current Protocols in Cell Biology 11.1.1-11.1.13., 1998 by JohnWiley & Sons, Inc. describes methods in vitro which can be used in thepresent invention.

According to the invention, advantageously, in the above labellingcompound (CI), n=1, and Y is a 3-pyridinyl group.

The compounds of formula (CI) may belong to various families, a firstfamily may be defined as that of the “alkyl ethers”, which correspond tothe following formula (CII):

in which p is an integer from 1 to 10, such as 2, 3, 4, 5, 6, 7, 8 or 9.

The preferred compounds of formula (CII) are chosen from the followingcompounds:

1-[(2-[¹⁸F]fluoropyridin-3-yloxy)methyl]pyrrole-2,5-dione

1-[2-(2-[¹⁸F]fluoropyridin-3-yloxy)ethyl]pyrrole-2,5-dione

1-[4-(2-[¹⁸F]fluoropyridin-3-yloxy)butyl]pyrrole-2,5-dione

1-[3-(2-[¹⁸F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione

1-[5-(2-[¹⁸F]fluoropyridin-3-yloxy)pentyl]pyrrole-2,5-dione

1-[6-(2-[¹⁸F]fluoropyridin-3-yloxy)hexyl]pyrrole-2,5-dione

A second family of compounds of formula (CI) may be defined as those ofthe “phenylalkyl ethers”, which correspond to the following formula(CIII):

in which q and r represent independently an integer from 0 to 10, suchas 0, 1, 2, 3, 4, 5, 6, 7, 8, 9.

The preferred compounds of formula (CIII) are chosen from the followingcompounds:

1-{4-[2-(2-[¹⁸F]fluoropyridin-3-yloxy)ethyl]phenyl}pyrrole-2,5-dione

1-[4-(2-[¹⁸F]fluoropyridin-3-yloxymethyl)phenyl]pyrrole-2,5-dione

1-[4-(2-[¹⁸F]fluoropyridin-3-yloxymethyl)benzyl]pyrrole-2,5-dione

A third family is that of the compounds which correspond to thefollowing formula (CIV):

in which s is an integer from 1 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9.

A preferred compound of formula (CIV) is the following compound:

1-[3-(6-[¹⁸F]fluoropyridin-3-yl)propyl]pyrrole-2,5-dione

A fourth family is that of the compounds which correspond to thefollowing formula (CV):

in which t is an integer from 0 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9and T is a group —CH═CH— or —C═C—.

Preferred compounds of formula (CV) are the following compounds:

1-[3-(6-[¹⁸F]fluoropyridin-3-yl)allyl]pyrrole-2,5-dione

1-[3-(6-[¹⁸F]fluoropyridin-3-yl)prop-2-ynyl]pyrrole-2,5-dione

The labelling compound (CI) may be prepared by a method in which:

a) a precursor compound of formula (CIa):

in which PR₁ and PR₂ represent independently a hydrogen atom or a groupprotecting the amine functional group, provided that PR₁ and PR₂ are notboth (simultaneously) a hydrogen atom, or alternatively PR₁ and PR₂together form with the nitrogen atom a cyclic group protecting the aminefunctional group, Gp represents a leaving group which can be replacedwith a fluorine-18 atom, and β, Y, m and n have the meanings alreadygiven above; is brought into contact with a source of fluoride ions F⁻labelled with [¹⁸F], to give a compound of formula (CIb):

b) the group(s) PR₁ and/or PR₂ protecting the amine functional group is(are) removed from the compound (Ib) to give a compound of formula (Ic):

c) the compound (CIc) is reacted with a reagent capable of giving amaleimido group from an amino group, so as to obtain the final compoundof formula (CI).

The method according to the invention is simple, reliable, easy to carryout and may be easily automated. It comprises only three steps in whichone is an extremely simple deprotection step.

The overall duration of the method is short: by way of example, it isgenerally from 60 to 120 minutes, preferably from 75 to 85 minutes.

The incorporation of the halogen fluorine-18 is carried out in anextremely efficient manner with a high yield, for example 70 to 100%, inparticular due to the fact that it is carried out on a heterocyclicgroup such as pyridine.

The final yield of the entire method for a purified product is extremelyhigh, for example from 15% to 25% and the potential quantities of the“synthon” compound, at the end of synthesis, are also very high.

In the compound (CIa), the groups PR₁ and PR₂, when they are protectivegroups, may be any protective group known in organic chemistry. They arepreferably chosen from the tert-butoxycarbonyl (BOC) andfluorenylmethoxycarbonyl (FMOC) groups.

When PR₁ and PR₂ together form with the nitrogen atom of the aminefunctional group, a group protecting the latter, the protecting groupmay be for example a phthalimido group.

In the compound (CIa), the Gp group may be any leaving group capable ofbeing replaced by a fluorine-18 atom; Gp is preferably chosen fromhalogens such as F, Cl, Br, I, mesyl, tosyl and triflate groups, when Yis an alkyl group; and Gp is preferably chosen from halogens, ammoniumsalts, such as trimethylammoniumtrifluoro-methanesulphonate, and thenitro group, when Y is an aromatic or heterocyclic group.

In step a), the source of fluoride ions labelled with ¹⁸F comprises thesaid fluoride ions and a counter-ion, chosen from large-sized cationssuch as rubidium, and tetrabutylammonium, and small-sized cations suchas potassium, sodium and lithium, the said small-sized cations beingtrapped, stabilized, for example by a cryptand or a crown ether, and thelike, the said cryptand or crown ether being suitable for thesmall-sized cation used.

An example of a cryptand is the product KRYPTOFIX® K₂₂₂:(4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexa-cosane) whichtraps for example the potassium ion.

The counter-ion or cation may be brought in the form of any salt, forexample it may be K₂CO₃, in the case of potassium.

Step a) is generally carried out in a solvent, which may be any suitablesolvent, such as DMSO.

Step a) may be carried out under conditions known to persons skilled inthe art, with heating generally at a temperature of 50 to 200° C., forexample, 145° C., for a period generally of 1 to 30 minutes, for exampleof 4 to 6 minutes.

Step b) for removing the group protecting the amine functional group,for deprotection, to give the compound of formula (CIc), where the aminogroup is free, may be carried out by any known deprotection method. Itwill be possible for example to bring the compound (CIb) into contactwith TFA in CH₂Cl₂ for a period generally of 1 to 5, for example of 2minutes.

It should be noted that TFA is generally used only if the receptor groupis removed in an acidic medium, for example when PR₁=BOC and PR₂=H.

In step c), the reagent capable of giving a maleimido group from anamido group may be any known compound. It may thus be chosen fromN-methoxycarbonylmaleimide and succinimide.

Step c) may be carried out under conditions known to persons skilled inthe art, for example in a solvent, such as xylene, THF, with heatinggenerally at a temperature of 100 to 200° C., for example of 190° C.,for a period of 1 to 20 minutes, for example of 5 minutes.

Step c) may, in another embodiment, also be carried out in a biphasicmixture for example of dioxane and aqueous sodium bicarbonate, at roomtemperature for a period of 3 to 15 minutes, for example 10 minutes;this embodiment of step c) offers the advantage of giving a better yieldand of being carried out at room temperature, without the need to heatthe mixture.

The compound of formula (CIa) may correspond to the following formula(CIIa):

The compound (CIIa) preferably corresponds to the following formula(CIIb):

The compound of formula (CIa) may, in another embodiment, correspond tothe following formula (CIIIa):

The compound (CIIIa) preferably corresponds to the following formula(CIIIb):

The compound of formula (CIa) may, in yet another embodiment, correspondto the following formula (CIVa):

The compound (CIVa) preferably corresponds to the following formula(CIVb):

In another embodiment, the compound of formula (CIa) may correspond tothe following formula (CVa):

The compound (CVa) preferably corresponds to the following formula(CVb):

The present invention also relates to a method for synthesizing thepeptide labelled with fluorine-18 in accordance with the presentinvention. This method of synthesis comprises a step of addition of acompound (CI) defined above with a peptide comprising the sequence (PI)defined above. This is indeed an addition reaction carried out betweenthe double bond of the maleimide functional group of the compound (CI)and a free —SH functional group of the peptide, in particular the thiolfunctional group of a cysteine, of the peptide comprising the peptidesequence (PI). The addition may be carried out directly on a free —SHfunctional group of the peptide sequence (PI), in particular on thethiol functional group of a cysteine of the peptide sequence, asdescribed above. This addition may be made for example in anacetonitrile/methanol solvent in a ratio of 2:1 by volume, respectively,or in any other appropriate solvent for this type of addition reaction.It will of course be necessary to take care that the solvent used doesnot affect the peptide (PI) of the invention.

This method therefore has the advantage of being easy to carry outunlike the labelling methods of the prior art.

The coupling will occur, while preserving the activity of the peptide ofthe present invention, and in general at the ends or at the level of theends of the peptide of the present invention, on surface residues, or ona part of the peptide sequence different from the sequence (PI) definedabove and in particular on the sequence (PII).

The present invention also provides a labelled assemblage havingaffinity for a phospholipid, comprising at least two peptides comprisingthe sequence (PI) defined above, which are identical or different, thesaid peptides being linked to each other, and each or only one of thesepeptides being labelled by means of a labelling compound (CI) accordingto the invention. These assemblages may be obtained for example byinserting a flexible peptide linkage, for example polyglycine, betweenthe C-terminal residue of a peptide of the invention and the N-terminalresidue of the second peptide and so on depending on the number ofpeptides joined end to end. This polyglycine linkage may be of formula-(Gly)_(n)-, n being an integer ranging from 1 to 12, for examplegreater than 4.

These assemblages may also be synthesized by conventional methods oforganic chemistry synthesis or protein chemistry, and by geneticrecombination in vivo or in vitro, by genetic engineering, and the like,for example by one of the abovementioned methods.

These assemblages are designed in particular to increase the affinity ofthe peptides of the present invention for the phospholipid, for examplefor a negatively charged phospholipid.

A labelled peptide or a labelled assemblage of the present invention maybe used in two ways which are research and diagnosis, and there arenumerous applications.

The pathologies especially targeted by the present invention are: (i)blood clotting disorders, (ii) the phenomena of apoptosis following theaction of chemical compounds, physical effects such as ionizingradiation, biological effects such as those linked to the formation orthe necrosis of cancer tissues, in addition to normal apoptosisphenomena, (iii) inflammatory pathologies, and (iv) disorders associatedwith the relationships between the cells and the extracellular matrixand in particular collagen.

The peptides of the present invention have in addition a great advantagecompared with the prior art compounds: the reversibility of theirfolding processes which allows their handling at high temperatures butwhich are compatible with the chemical stability of the peptides, forthe purposes of chemical modifications with the aim of developingmolecules which can be used in imagining.

In addition, because of their small size, the peptides of the presentinvention may be easily combined with other proteins either to formmultifunctional chimeric proteins, or to introduce a mechanism forregulation by effectors other than the signalling phospholipids.

According to the invention, the peptides and assemblages according tothe invention coupled to the compound (CI) form labelling compoundswhich can be used for example for in vivo or in vitro diagnosis.

Indeed, the peptides of the present invention may be used for thedetection of pathologies involving the appearance of negative charges atthe surface of cells and the release of microvesicles into the blood:for example clotting disorders, acute inflammatory pathologies and thelike, and apoptosis.

The radioactive halogen is fluorine-18 which is a radio element with ashort life span because it allows “in vivo” detection of thelocalization of the thrombotic regions during all sorts of stroke, inparticular of the apoptotic and inflammatory foci using appropriateimaging systems.

The peptides or assemblages labelled with fluorine-18, according to thedesired application, may be advantageously packaged in the form ofdiagnostic kits. Thus, the present invention also provides a diagnostickit comprising a labelled peptide or assemblage in accordance with thepresent invention.

The present invention also provides a kit for the analysis and detectionof negative charges at the surface of cells, characterized in that itcomprises a labelled peptide or assemblage of the present invention.

The present invention also provides a kit for the analysis and detectionof microvesicles in the blood, characterized in that it comprises alabelled peptide or assemblage in accordance with the present invention.

The peptides labelled with fluorine-18 according to the invention cantherefore be used for the manufacture of a product intended for thedetection of centres exposing negatively charged lipids at the surfaceof cells and/or the release of microvesicles into the blood. Asspecified above, the detection may be a detection by means ofscintigraphic images acquired by positron emission tomography, becausethe compound (CI) comprises ¹⁸F.

In their application, in the context of “PET”, the compounds (CI) andthe labelled peptides according to the invention, comprising afluorine-18 atom, show numerous advantages compared with the compoundswith another radioactive halogen, for example iodine.

Indeed, the only positron-emitting iodine isotope is iodine-124,which-could allow PET.

However, it is still produced in small quantities (a few mCi againstcuries for F-18). It is also difficult to produce. Finally, iodine-124is not a pure positron emitter (fluorine-18, 97%) and decreases by beta+emission at 25% only and by electron capture at 75%; it possesses alarge number of gamma lines ranging from 0.603 MeV (62%) to 2.75 MeV(1%).

The invention additionally relates to compositions for analysis anddetection for example by positron emission tomography (PET), orcompositions for diagnosis comprising a peptide labelled withfluorine-18 as described above and a pharmaceutically acceptablevehicle.

Other advantages and characteristics of the present invention willfurther emerge on reading the illustrative and nonlimiting exampleswhich follow, with reference to the figures in the annex.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The appended sequences ID No. 1 to ID No. 14 are examples of peptidescontaining the peptide sequences (PI) and (PII) of the presentinvention.

In particular, the sequences ID No. 11, ID No. 13 and ID No. 14 areexamples of peptides containing the peptide sequence of the presentinvention in which mutations have been introduced in order to increasethe affinity for calcium and phospholipids.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2 are micrographs obtained from tissue sections of anapoptotic heart (FIG. 1) and of a kidney (FIG. 2), respectively. Thesesections were obtained, on the one hand, (photos on the left) withAFIM-fluorescein (AFIM-F) peptides of the present invention, on theother hand (photos on the right) with annexin 5-fluorescein (A5-F)(compound of the prior art): fluorescence microscopy, magnification ×40.The photos in the centre were obtained with haematoxylin: visible lightmicroscopy, magnification ×40. In FIG. 1, the top and bottom photosrepresent different heart sections.

FIG. 3 is a graph which represents the degree of helicity “H” (in %) ofa peptide according to the present invention as a function of thetemperature “t” in ° C.

EXAMPLES Example 1 Synthesis by Genetic Recombination: Expression andPurification of the Peptides of Sequences ID No. 1 to ID No. 12 of thePresent Invention

The sequences ID No. 1 to ID No. 14 were prepared by overexpression inE. coli according to the same protocol as that which has been describedby F. Cordier-Ochsenbein et al. in J. Mol. Biol. 279, 1177-1185.

The cDNAs of each of these sequences were prepared using a polymerasechain reaction (PCR). They were inserted into the vector pGEX-2T (Smith& Johnson, 1998). FIG. 2 is an image illustrating the insertion of thecDNA into the vector. The absence of PCR-induced mutations was checkedby sequencing.

The production of the peptides is carried out using the strain E. coliBL21 containing the expression vector described above. After inductionwith isopropylthiogalactopyranoside (IPTG, 100 μM) to an optical densityof 1 to 600 nm, the growth is continued until a plateau is reached, thatis to say for about 3 hours. After centrifugation, the bacteria areresuspended in the lysis buffer comprising 50 mM Tris-HCl, pH 8, 10 mMEDTA, 500 mM NaCl, 5% (v/v) glycerol, 1% (v/v) Triton X100, 1 mMdithiothreitol (DTT), 1 mM phenylmethylsulphonyl fluoride (PMSF) and 20μg/ml of aprotinin.

The purification was carried out in the following manner: aftersonication and centrifugation at 10 000 g, the supernatant containingthe soluble proteins is incubated with glutathione/agarose beadsallowing specific binding of the GST-domain fusion protein to thesebeads. After washing with a solution containing 1 M NaCl, 50 mM Tris-HClat pH 8, 70 units of thrombin per litre of culture are added and thesequences are eluted.

The sequences are then purified on a proRPC (trademark) column of the16/10 type, supplied by the company Pharmacia using an FPLC system and alinear gradient of water of Millipore (trademark) quality containing0.1% (v/v) of trifluoroacetic acid TFA, and acetonitrile containing 0.1%of TFA. The flow rate is adjusted to 2.5 ml/minute. The sequences arethen freeze-dried.

The final yield for each peptide is about 8 mg of sequence per litre ofculture.

Example 2 Example of Chemical Synthesis of Peptides of the PresentInvention

The peptides of the present invention were manufactured in this exampleby solid phase chemical synthesis with an Applied Biosystems, mod. 433Aautomatic peptide synthesizer, and by Fmoc chemistry, which uses thefluorenylmethyloxycarbonyl (Fmoc) group for the temporary protection ofthe α-amino functional group of the amino acids.

The protecting groups used to prevent side reactions of the side chainsof amino acids, in this strategy Fmoc, were tert-butyl ether (tBu) forthe Ser, Thr and Tyr residues; tert-butyl ester (OtBu) for Asp, Glu;trityl (Trt) for Gln, Asn, Cys, His; tert-butyloxycarbonyl (Boc) for Lysand 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc) for Arg.

The coupling reaction is carried out with an excess of 10 equivalents ofamino acids (1 mmol) relative to the resin (0.1 mmol). The protectedamino acid is dissolved in 1 ml of N-methylpyrrolidone (NMP) and 1 ml ofa 1M solution of 1-N-hydroxy-7-azabenzotriazole (HOAt) in the solventNMP. 1 ml of a 1M solution of N,N′-dicyclohexylcarbodiimide (DCC) isthen added. After 40 to 50 minutes of activation, the active esterformed is transferred into the reactor which contains the resin. Beforethis step of transfer and then of coupling, the resin is deprotected ofits Fmoc group by a 20% solution of piperidine in NMP. The excesspiperidine is removed by washing with NMP after about 5 to 10 minutes.

During the deprotection, the detection of the dibenzofulvenepiperidineadducts at 305 nm makes it possible to monitor the good progress of thesynthesis. Indeed, the quantification of the adduct makes it possible toestimate the efficiency of the deprotection of the Fmoc group andthereby of the coupling of the last amino acid incorporated.

The cleavage of the resin and of the protecting groups present on theside chains was carried out simultaneously by treating the peptidelinked to the resin with trifluoroacetic acid (TFA). Before carrying outthe cleavage, the resin was washed several times with dichloromethane(DCM) and finally dried. The reagent used during the cleavage is an acidmixture containing 81.5% of TFA and the phenol scavengers (5%), water(5%), ethanedithiol (2.5% when the peptide contains a cysteine) andtriisopropylsilane (1%). The resin was treated with this mixture forthree hours, with stirring and at room temperature, in an amount of 100ml of solution per gram of resin. The free peptide in solution wasrecovered by filtration. The peptide was then precipitated and washed inthe cold state in diisopropyl ether and then dissolved in 20% aceticacid and freeze-dried.

The peptide recovered after freeze-drying, the crude material fromsynthesis, is in reduced form, that is to say that the interchaindisulphide bridges are not formed.

The peptide is then purified on a proRPC (trademark) column of the 16/10type, supplied by the company Pharmacia using an FPLC system and alinear gradient of water of Millipore (trademark) quality containing0.1% by volume of trifluoroacetic acid TFA, and acetonitrile containing0.1% of TFA. The flow rate is adjusted to 2.5 ml/minute. The peptide isthen freeze-dried.

The products obtained were analysed by mass spectrometry.

Example 3 Stability of the Sequences ID No. 1 to ID No. 14

This example shows that the peptides of the present invention constitutestable folding proteins.

Composition of the Blank (Control): Tris 50 mM, NaCl 150 mM, DTT 1 mM pH8  10 μl H₂O 990 μl Adjusted to pH 8Composition of the Sample:

Sample: domain purified in 50 mM Tris buffer containing 150 mM NaCl, pH8 Approx. concentration: 200 mg.ml.

Domain: 10 μl that is 300 μM final.

H₂O: 990 μl

pH measured at 7.8.

Hardware and Software Configuration:

Apparatus Jobin Yvon CD6.

Software CD-max

Optical path length of the measurement cuvette: 1 cm.

The appended FIG. 1 represents the degree of helicity of AFIM as afunction of the temperature as measured with the aid of the circulardichroism signal in far UV at the wavelength of 220 nm.

In this figure, the value of the signal at 14° C. is taken as 100% ofthe helical content of the peptide. Heat denaturation of the peptide isindeed cooperative and demonstrates that at low temperature and inparticular at 37° C., this is a peptide which is suitably folded andexhibiting enhanced stability.

Example 4 Assemblages of Two Peptides of the Present Invention

The method described in Example 1 above is used to synthesize a peptidesequence with the sequence ID No. 1-(gly)₄-ID No. 1.

The final yield for the assemblage is about 14 mg/litre of culture.

This assemblage may be labelled with a radioactive halogen according tothe present invention, in the same manner as the peptide alone, forexample by the method described below.

Example 5 Synthesis of a Labelling Compound of the Present Invention

In this example, there is described the preparation of a labellingcompound according to the invention, which is1-[3-(2-[¹⁸F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione.

a) Complex K[¹⁸F]F-K₂₂₂.

In order to recover and recycle the water target [¹⁸O ], it is caused topass through an anion-exchange resin (AG1×8, from Bio-Rad, 100-200mesh). The fluoride [¹⁸F] ion is then eluted from the resin, using 1.0ml of an aqueous solution of K₂CO₃ at 4.5 mg/ml.

After addition of 11.0 to 15.0 mg of KRYPTOFIX® K₂₂₂(4,7,13,16,21,24-hexaoxa-1,10-diazobicyclo[8.8.8]hexa-cosane), theresulting solution is then gently concentrated to dryness at 145-150°C., under a nitrogen stream for 10 minutes in order to give a pureK[¹⁸F]F—K₂₂₂ complex, in the form of a white semisolid residue.

b) 1-[3-(2-[¹⁸F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione

Freshly distilled DMSO (600 μl), containing 4.0 to 6.0 mg of the “nitro”marker precursor (tert-butyl ester of[3-(2-nitropyridin-3-yloxy)propyl]carbamic acid) is added directly tothe tube containing the dried K[¹⁸F]—K₂₂₂ complex. The tube (not sealed)is then placed in a heating block (at 145° C. for 4 minutes). The tubeis then cooled using an ice/water bath and the remaining radioactivityis measured.

85% to 95% of the initial activity placed in the container is stillpresent. The reaction mixture obtained, which is dark in colour, is thenanalysed by radiochromatography. The incorporation yields are calculatedfrom the radiochromatogram by TLC and are defined by the ratio of thesurface of the tert-butyl ester of[3-(2-[¹⁸F]fluoropyridin-3-yloxy)propyl]carbamic acid to the totalactivity of the ¹⁸F fluorine-18 (SiO₂-TLC; eluent: EtOAc; Rf: Rf: 0.75and Rf: fluoride [¹⁸F] ion: 0.0). The reaction mixture is diluted with 1ml of water and transferred into a C18 Sep-pak cartridge (Waters). Thetube is rinsed twice with 1 ml of water, which is also transferred andadded to the dilute reaction mixture in the cartridge.

The whole is then caused to pass through the cartridge. The cartridge iswashed with 3 ml of water and partially dried for 0.5 minute, by sendinga nitrogen stream.

The tert-butyl ester derivative of[3-(2-[¹⁸F]fluoro-pyridin-3-yloxy)propyl]carbamic acid is eluted fromthe cartridge with 3 ml of dichloromethane in a reaction flaskcontaining 0.1 ml of TFA. Twice 1 ml of dichloromethane are used to washthe cartridge and to completely transfer the [¹⁸F]-labelled derivativementioned above (5% of the total quantity of radioactivity involved inthe fluorination process remains on the cartridge). The incorporationyield is also confirmed after elution of the Sep-pak by the ratio of thecount values for CH₂Cl₂ to the total radioactivity eluted(DMSO/H₂O+CH₂Cl₂). The resulting CH₂Cl₂/TFA solution (50/1, V/V) isconcentrated to dryness (at 65-75° C.) under a moderate nitrogen streamfor 4 to 6 minutes). The deprotection yield is quantitative: no moleculedescribed above, protected with BOC, can be detected byradiochromatography. The above residue is redissolved in 2 ml of CH₂Cl₂and again concentrated to dryness in order to minimize the presence ofTFA (at 65-75° C. under a moderate nitrogen stream for 4 to 6 minutes).The residue is then diluted with 0.5 ml of xylene containing 25 mg ofN-methoxycarbonylmaleimide. The container is then hermetically closed,heated for 5 minutes at 190° C. (strong reflux), and then cooled for 2minutes, using an ice-water bath. The reaction mixture is then injectedonto a semipreparative HPLC column. Isocratic elution [eluent:heptane/EtOAc: 50/50; flow rate 6.0 ml/minute] which gives pure labelled1-[3-(2-[¹⁸F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione, retentiontime: 7.5 to 8.0 minutes.

Typically, 60 to 70 mCi of pure labelled1-[3-(2-[¹⁸F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione may beobtained in 75 to 85 minutes, from 550-650 mCi from an [¹⁸F]F⁻production batch of a cyclotron.

Example 5a

The compound labelled with fluorine-18,1-[3-(2-[¹⁸F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione may also beprepared by repeating steps a) and b) of the method described in Example5, still using, as labelling precursor, the “nitro” compound (tert-butylester of [3-(2-nitropyridin-3-yloxy)propyl]carbamic acid), but modifyingthe final part of the preparation (step c)) in the following manner(variant according to which step c) is carried out in a biphasic mixtureof dioxane and aqueous sodium bicarbonate).

After deprotection of the amine functional group (TFA/CH₂Cl₂), theresidue obtained after concentration to dryness is taken up in 0.250 mlof dioxane containing 25 mg of N-methoxycarbonylmaleimide. To thissolution, 0.750 ml of a saturated aqueous sodium bicarbonate solution isadded, and the preparation is vortexed at room temperature for 10minutes. The reaction mixture is then diluted with 1 ml of water andtransferred onto a C18 Sep-pak cartridge (Waters). The flask is rinsedtwice with 1 ml of water, which is also transferred and added to thedilute reaction mixture in the cartridge. Finally, 8 ml of water areagain added to the dilute reaction mixture in the cartridge. The wholeis then passed through the cartridge. The cartridge is washed with 3 mlof water and partially dried for 0.5 minutes, by sending a nitrogenstream. The derivative labelled with fluorine-18(1-[3-(2-[¹⁸F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione) is elutedfrom the cartridge with 3 ml of dichloromethane in a new empty flask. 1ml of dichloromethane is used twice to wash the cartridge and tocompletely transfer the [¹⁸F]-labelled derivative mentioned above. Thesolution containing the abovementioned [¹⁸F]-labelled derivative isconcentrated (at 65-75° C., under a moderate nitrogen stream for 3 to 5minutes) to a volume of about 1 ml and injected onto a semipreparativeHPLC column. The purification is identical to that described in Example5.

Example 5b

The compound labelled with fluorine-18,1-[3-(2-[¹⁸F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione may also beprepared by repeating steps a) and b) of the method described in Example5 or 5 b, but using, as labelling precursor, the compound“trimethylammonium trifluoromethanesulphonate”([3-(3-tert-butoxycarbonyl-aminopropoxy)pyridine-2-yl]trimethylammoniumtrifluoro-methanesulphonate).

Example 6 Labelling of a Peptide of the Present Invention withFluorescein

This example, as well as Example 7 which follows, are intended todemonstrate the efficiency of recognition of apoptotic sites by thepeptides of the present invention.

In the examples which follow, the peptide of the present invention iscalled AFIM-SH. It has a peptide sequence as defined by the sequence(PI). The sequences ID No. 1 to ID No. 14 are tested.

Fluorescein is a molecule which emits a green fluorescence having awavelength of 525 nm when it is excited at a wavelength of 488 nm. Theemission of green light is detected by cameras or photomultipliers. Thiscoupling of AFIM to fluorescein makes it possible to detect the presenceof the cells exhibiting PS both in vitro and in vivo in small animals.

According to the present invention, it is possible to label AFIM at thelevel of the surface residues on any cystein which would be introducedin place of any amino acid present at the surface of AFIM (surfaceresidues) as long as the function for binding to the lipid membranes isnot disrupted. AFIM thus modified is designated AFIM-SH below.

The coupling of fluorescein occurs via a maleimide functional grouprepresented below on AFIM by the SH functional group.

The fluorescein is covalently coupled to one or more cysteins of thesequence using a maleimide functional group.

The entire labelling is performed at a temperature of less than 20° C.

AFIM-SH is in solution in Tris buffer. (50 mM), containing NaCl (150mM), pH=7.4. 5 equivalents of DTT in solution in the same buffer areadded to the AFIM-SH solution. The medium is stirred for 30 min.

Protected from light: the fluorescein (5 equivalents of AFIM-SH+2equivalents of DTT) is weighed and dissolved in DMF, and added to thepreceding solution. The whole is stirred, and the reaction is continuedfor 30 min. Next, the medium is diluted in 150 ml of PBS buffer (20 mMphosphate, 150 mM NaCl), pH=7.4, and ultrafiltered on YM3 (trademark)membrane. The sample is rediluted and ultrafiltered several times,determining the UV spectrum of the filtrate.

When there is no longer any fluorescein in the filtrate (peak at 490nm), the sample is concentrated to a few ml and stored in the cold at 4°C.

The AFIM-fluorescein products were used to detect apoptotic cells byflow cytometry in vitro, and in animals in vivo in the manner describedin Example 7 which follows.

Example 7 Results for Labellings of Apoptotic Cells with theAFIM-fluorescein Products of Example 6

Imaging of apoptotic cardiac cells following a heart attack in rats.

A model of apoptosis in rats is used as described in the article whichappeared in Circulation Res. 1996, 79, 946-956.

Briefly, four rats (300 g each) were anaesthetized, intubated andventilated. Myocardial ischaemia was triggered by a transient occlusionof the coronary artery. After 30 minutes of occlusion, the coronaryartery was reperfused for one hour.

At the end of the reperfusion period, the AFIM-fluorescein peptides ofExample 6 were injected into the jugular vein in an amount of 200 μg ofpeptide for each of two of the rats in a total volume of 1 ml.

By way of comparison, 200 μg of annexin 5-fluorescein (compounds of theprior art) were injected under the same conditions for each of the othertwo rats in a total volume of 1 ml.

The rats were sacrificed after 60 minutes.

Five organs were stored for this study: the heart, the lung, the kidney,the liver and the brain. They were washed and rinsed in the presence offormalin. The organs were then dehydrated and impregnated with paraffinfor about 12 hours and then 7 μm sections were prepared.

A few sections were stained with haematoxylin. The sections wereexamined under a fluorescence microscope and the adjacent sectionsstained with haematoxylin were examined with a visible light microscope.The sections stained with haematoxylin (labelled H1 and H2 respectivelyin the appended FIGS. 1 and 2) allow visualisation of the architectureof the tissues and fluorescence microscopy to detect the labelling withAFIM-fluorescein (AFIM-F) or with annexin 5-fluorescein (A5-F).

The appended FIG. 1 shows the images obtained for the apoptotic heartand the appended FIG. 2 shows the images obtained for the kidney.

FIG. 1 clearly shows the excess of fluorescence corresponding to theaccumulation of marker at the level of the apoptotic cells. The contrastis visibly better with AFIM of the present invention than with the priorart annexin 5.

FIG. 2 shows the labelling of the kidney linked to the partialelimination of the products. In the case of AFIM, the glomeruli do notappear to be labelled, only the proximal tubules are partially labelled.On the other hand, in the case of the prior art annexin 5, the entirerenal tissue is strongly labelled, which is in agreement with the renaltoxicity observed for this protein.

The results obtained in this example demonstrate a high specificity ofthe peptides of the present invention for the labelling of the cells.

The labelling of the AFIM peptide, for example from ID No. 1 to 10, byfluorescein therefore makes it possible to efficiently detect thephosphatidylserine (PS) present at the outer surface of the cellsinvolved in physiopathological processes such as programmed cell death(apoptosis), blood clotting, inflammatory reaction.

Example 8 Labelling According to the Method of the Present Invention ofPeptides Comprising the Sequence (PII) with the Labelling Compound (CI)

In the examples which follow, the peptide of the present invention iscalled AFIM-SH. It has a peptide sequence as defined by the sequence(PII). The sequences ID No. 1 to ID No. 14 of the appended sequencelisting are tested. The labelling compound called synthon ¹⁸Fmanufactured in Example 5 (or 5a or 5b) is used in this example.

AFIM is coupled, specifically at the level of an SH functional group ofthe cystein J^(□) to the synthon ¹⁸F.

The general scheme for the labelling may be summarized in the followingmanner:

AFIM-SH is in solution in Tris buffer (50 mM) containing NaCl (150 mM),pH=7.4. The synthon ¹⁸F is dissolved in an acetonitrile-methanol (2/1v/v) mixture, and AFIM-SH is added. The whole is stirred, and thereaction is continued for 3 minutes at room temperature.

The reaction medium is then transferred onto a column of maleimide beadssuspended in DMF, and eluted with PBS buffer.

The medium is purified by HPLC on an exclusion gel column, and eluted inPBS buffer (20 mM KH₂PO₄, 150 mM NaCl, pH=7.4).

The product, once purified is intravenously injected into rats.

1. Peptide labelled with fluorine-18, characterized in that it comprises the following peptide sequence (PI): J¹-J²-J³-J⁴-J⁵-J⁶-Z⁷-U⁸-J⁹-J¹⁰-U¹¹-Arg-J¹³-J¹⁴- U¹⁵-Lys-Gly-X¹⁸-Gly-Thr-J²¹-Glu-J²³-J²⁴-U²⁵-J²⁶- J²⁷-J²⁸-U²⁹-J³⁰-J³¹-Arg-J³³-J³⁴-J³⁵-J³⁶-B³⁷-J³⁸- J³⁹-U⁴⁰-J⁴¹-J⁴²-J⁴³-U⁴⁴-J⁴⁵-J⁴⁶-J⁴⁷-J⁴⁸-J⁴⁹-Arg- J⁵¹-U⁵²-J⁵³-J⁵⁴-Asp-U⁵⁶-Lys-Ser-Z⁵⁹-Leu-J⁶¹-J⁶²- J⁶³-J⁶⁴-Z⁶⁵-J⁶⁶-J⁶⁷-U⁶⁸-J⁶⁹-J⁷⁰-J⁷¹-U⁷²-J⁷³-J⁷⁴- J⁷⁵ (I)

in which J, Z, U, X and B represent amino acids such that: the amino acids J are chosen independently of each other from natural amino acids, or derivatives thereof, in such a manner that at least 50% of them are polar residues chosen from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Lys, Orn, Pro, Ser, Thr and Tyr, the amino acids U are chosen from Ala, Cys, Gly, Ile, Leu, Met, Phe, Trp, Tyr and Val, the amino acid X¹⁸ is chosen independently of the other amino acids of the sequence from Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Ser, Thr, Trp, Tyr and Val, the amino acid B³⁷ is chosen independently of the other amino acids of the sequence from Arg, Ala, Cys, Gly, Ile, Leu, Met, Phe, Trp, Tyr and Val, the amino acid Z⁷ is chosen independently of the other amino acids of the sequence from Asp and Glu, the amino acids Z⁵⁹ and Z⁶⁵ are chosen independently from Glu, Asp, Lys and Arg, the superscripts of J, Z, U, X and B representing the positions of these amino acids in the said sequence, the said peptide being labelled directly or indirectly with a compound (CI) of general formula:

in which: m represents an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n represents an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Y represents a group chosen from alkyl groups, monocyclic or bicyclic heterocyclic groups chosen from imidazolyl, pyrazolyl, benzimidazolyl, pyridinyl, piridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl and purinyl groups, it being possible for Y to be optionally substituted with one or more substituents, each of these substituents being chosen independently from hydrogen, (nonradioactive) halogens, phenyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, aryloxy, amino, mono- or di(C₁₋₆ alkyl)amino, mono- or di(aryl)amino, thio, C₁₋₆ alkylthio, arylthio, formyl, C₁₋₆ alkylcarbonyl, arylcarbonyl, carbonyl, C₁₋₆ alkoxycarbonyl, aryloxycarbonyl, C₁₋₆ alkylaminocarbonyl, arylaminocarbonyl and trifluoromethyl groups; β represents a radical of formula: (γ)_(a)-((CR₁R₂)_(b)—(V)_(c))_(d)—((CR₃R₄)_(e)—(W)_(f))_(g)— in which: a, b, c, d, e, f, g each independently represent an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9; γ, V and W each independently represent —NR—₁, —O—, —S—,

ethynyl, —CR₁═CR₂, —(C═O)—, —(C═S)—, —C(═NR₁)—, —C(═O)O—, —(C═S)S—, —C(═NR₁)NR₂—, —CR₁R₂—, —CR₁OR₂—, —CR₁NR₂R₃—, where R₁, R₂, R₃ and R₄ are independently chosen from hydrogen, halogens, phenyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, aryloxy, amino, mono- or di(C₁₋₆ alkyl)amino, mono- or di(aryl)amino, thio, C₁₋₆ alkylthio, arylthio, formyl, C₁₋₆ alkylcarbonyl, arylcarbonyl, carbonyl (C₁₋₆)alkoxycarbonyl, aryloxycarbonyl, C₁₋₆ alkylaminocarbonyl, arylaminocarbonyl and trifluoromethyl groups, directly or indirectly on an —SH functional group.
 2. Peptide labelled with fluorine-18 according to claim 1, in which the amino acids J are chosen independently of each other from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val in such a manner that at least 50% of them are polar residues chosen from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Lys, Pro, Ser and Thr.
 3. Peptide labelled with fluorine-18 according to claim 1, in which the amino acids U and B of the sequence (PI) are chosen according to one of Examples a) to j) presented in Table 1 below: U⁸ U¹¹ U¹⁵ U²⁵ U²⁹ B³⁷ U⁴⁰ U⁴⁴ U⁵² U⁵⁶ U⁶⁸ U⁷² Ex a) Val Leu Met Ile Leu Arg Ile Tyr Leu Leu Val Leu Ex b) Ala Ile Ile Ile Leu Arg Ile Tyr Leu Leu Ile Leu Ex c) Ala Ile Ile Ile Leu Arg Ile Tyr Leu Leu Met Val Ex d) Ala Leu Met Leu Leu Arg Ile Tyr Leu Leu Ile Met Ex e) Ala Leu Met Ile Ile Arg Val Tyr Leu Leu Ile Met Ex f) Ala Leu Met Ile Ile Arg Ile Phe Leu Leu Ile Met Ex g) Ala Leu Met Ile Val Arg Ile Phe Leu Leu Ile Phe Ex h) Val Leu Met Ile Leu Arg Ile Phe Leu Leu Ile Met Ex i) Ala Leu Met Ile Leu Arg Ile Phe Leu Leu Ile Met Ex j) Ala Leu Met Ile Leu Arg Ile Tyr Leu Leu Ala Ala (Ex = exemple)

(Ex=Example)
 4. Peptide labelled with fluorine-18 according to claim 1, in which the peptide sequence is chosen from the sequence ID No. 1, ID No. 2, ID No. 3, ID No. 4, ID No. 5, ID No. 6, ID No. 7, ID No. 8, ID No. 9, ID No. 10, ID No. 11, ID No. 12, ID No. 13 and ID No. 14 of the appended sequence listing.
 5. Peptide labelled with fluorine-18 according to any one of claims 1 to 4, additionally comprising, linked to its N-terminal end, the amino acid sequence chosen from Gly-Ser-Cys and Gly-Cys-Ser.
 6. Peptide labelled with fluorine-18 according to any one of claims 1 to 4, additionally comprising, linked to its N-terminal end, an amino acid sequence chosen from Gly-Ser-Gly-Cys, Gly-Cys-Gly-Ser and Gly-Cys-Gly-Cys.
 7. Peptide labelled with fluorine-18 according to any one of claims 1 to 6, in which the peptide is labelled directly with the compound (CI) by coupling the maleimide functional group of the compound (CI) with a free —SH functional group of the said peptide, for example the thiol functional group of a cystein of the peptide.
 8. Peptide labelled with fluorine-18 according to any one of claims 1 to 6, in which the peptide is labelled directly with the compound (CI) by coupling the maleimide functional group of the compound (CI) with a free —SH functional group of the peptide sequence (PI), for example the thiol functional group of a cystein of the peptide sequence.
 9. Peptide labelled with fluorine-18 according to any one of claims 1 to 6, in which, in the compound of formula (CI), n=1, and Y is a 3-pyridinyl group.
 10. Peptide labelled with fluorine-18 according to claim 9, in which the compound (CI) corresponds to the following formula (CII):

in which p is an integer from 1 to 10, such as 2, 3, 4, 5, 6, 7, 8 or
 9. 11. Peptide labelled with fluorine-18 according to claim 10, in which the compound of formula (CII) is chosen from: 1-[2-(2-[¹⁸F]fluoropyridin-3-yloxy)ethyl]pyrrole-2,5-dione; 1-[4-(2-[¹⁸F]fluoropyridin-3-yloxy)butyl]pyrrole-2,5-dione; 1-[5-(2-[¹⁸F]fluoropyridin-3-yloxy)pentyl]pyrrole-2,5-dione; 1-[6-(2-[¹⁸F]fluoropyridin-3-yloxy)hexyl]pyrrole-2,5-dione; 1-[(2-[¹⁸F]fluoropyridin-3-yloxy)methyl]pyrrole-2,5-dione; 1-[3-(2-[¹⁸F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione.
 12. Peptide labelled with fluorine-18 according to claim 9, in which the compound of formula (CI) corresponds to the following formula (CIII):

in which q and r represent independently an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8,
 9. 13. Peptide labelled with fluorine-18 -according to claim 12, in which the compound of the formula (CIII) is chosen from: 1-{4-[2-(2-[¹⁸F]fluoropyridin-3-yloxy)ethyl]phenyl}pyrrole-2,5-dione; 1-[4-(2-[¹⁸F]fluoropyridin-3-yloxymethyl)phenyl]pyrrole-2,5-dione; 1-[4-(2-[¹⁸F]fluoropyridin-3-yloxymethyl)benzyl]pyrrole-2,5-dione.
 14. Peptide labelled with fluorine-18 according to claim 9, in which the compound of formula (CI) corresponds to the following formula (CIV):


15. Peptide labelled with fluorine-18 according to claim 14, in which the compound of formula (CIV) is 1-[3-(6-[¹⁸F]fluoropyridin-3-yl)propyl]pyrrole-2,5-dione.
 16. Peptide labelled with fluorine-18 according to claim 9, in which the compound of formula (CI) corresponds to the following formula (CV):

in which t is an integer from 0 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 and T is a group —CH═CH—or —C≡C—.
 17. Peptide labelled with fluorine-18 according to claim 16, in which the compound (CV) is chosen from: 1-[3-(6-[¹⁸F]fluoropyridin-3-yl)allyl]pyrrole-2,5-dione; 1-[3-(6-[¹⁸F]fluoropyridin-3-yl)prop-2-ynyl]pyrrole-2,5-dione.
 18. Peptide labelled with fluorine-18 according to any one of claims 1 to 6, in which the peptide sequence is chosen from the sequence ID No. 1, ID No. 2, ID No. 3, ID No. 4, ID No. 5, ID No. 6, ID No. 7, ID No. 8, ID No. 9, ID No. 10, ID No. 11, ID No. 12, ID No. 13 and ID No. 14 of the appended sequence listing, in which the compound (CI) is chosen from: 1-[3-(6-[¹⁸F]fluoropyridin-3-yl)allyl]pyrrole-2,5-dione; 1-[3-(6-[¹⁸F]fluoropyridin-3-yl)prop-2-ynyl]pyrrole-2,5-dione.
 19. Method for synthesizing a peptide labelled with a radioactive halogen according to any one of claims 1 to 6, comprising a step for adding a compound (CI) of general formula:

in which: m represents an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n represents an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Y represents a group chosen from alkyl groups, monocyclic or bicyclic heterocyclic groups chosen from imidazolyl, pyrazolyl, benzimidazolyl, pyridinyl, piridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl and purinyl groups, it being possible for Y to be optionally substituted with one or more substituents, each of these substituents being chosen independently from hydrogen, halogens, phenyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, aryloxy, amino, mono- or di(C₁₋₆ alkyl)amino, mono- or di(aryl)amino, thio, C₁₋₆ alkylthio, arylthio, formyl, C₁₋₆ alkylcarbonyl, arylcarbonyl, carbonyl, C₁₋₆ alkoxycarbonyl, aryloxycarbonyl, C₁₋₆ alkylaminocarbonyl, arylaminocarbonyl and trifluoromethyl groups; β represents a radical of formula: (γ)_(a)-((CR₁R₂)_(b)—(V)_(c))_(d)—((CR₃R₄)_(e)—(W)_(f))_(g)— in which: a, b, c, d, e, f, g each independently represent an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9; γ, V and W each independently represent —NR—₁, —O—, —S—,

ethynyl, —CR₁═CR₂, —(C═O)—, —(C═S)—, —C(═NR₁)—, —C(═O)O—, —(C═S)S—, —C(═NR₁)NR₂—, —CR₁R₂—, —CR₁OR₂—, —CR₁NR₂R₃—, where R₁, R₂, R₃ and R₄ are independently chosen from hydrogen, halogens, phenyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, aryloxy, amino, mono- or di(C₁₋₆ alkyl)amino, mono- or di(aryl)amino, thio, C₁₋₆ alkylthio, arylthio, formyl, C₁₋₆ alkylcarbonyl, arylcarbonyl, carbonyl (C₁₋₆)alkoxycarbonyl, aryloxycarbonyl, C₁₋₆ alkylaminocarbonyl, arylaminocarbonyl and trifluoromethyl groups; directly or indirectly onto an —SH functional group of a peptide.
 20. Method according to claim 19, in which the addition is carried out directly onto a free —SH functional group of the peptide sequence (PI), for example the thiol functional group of a cystein of the peptide sequence.
 21. Kit for analysis and detection of negative charges at the surface of cells, characterized in that it comprises a peptide labelled with fluorine-18 according to any one of claims 1 to
 18. 22. Diagnostic kit comprising a peptide labelled with fluorine-18 according to any one of claims 1 to
 18. 23. Kit for analysis and detection of microvesicles in blood, characterized in that it comprises a peptide labelled with fluorine-18 according to any one of claims 1 to
 18. 24. Use of a peptide labelled with fluorine-18 according to any one of claims 1 to 18 for the manufacture of a product intended for the detection of centres exposing negatively charged lipids at the surface of cells and/or the release of microvesicles into the blood.
 25. Use according to claim 24, in which the detection is a detection by means of scintigraphic images acquired by positron emission tomography (PET).
 26. Composition for analysis and detection for example by positron emission tomography (PET) having a peptide labelled with fluorine-18 according to any one of claims 1 to 18 and a pharmaceutically acceptable vehicle.
 27. Composition for diagnosis, comprising a peptide labelled with fluorine-18 according to any one of claims 1 to 18 and a pharmaceutically acceptable vehicle. 